Project Summary Iron is an essential element that is required in nearly all living organisms. Due to its gas binding and redox properties it plays an irreplaceable role in human physiology helping to facilitate the reactions necessary to sustain life. Ferroportin (FPN) acts as the sole iron efflux protein in humans, and all iron transport to the plasma occurs through this single channel. Thus, FPN plays a critical role in the maintenance of human iron homeostasis. The regulatory hormone hepcidin controls the levels of membrane associated FPN through the induction of endocytosis and proteasomal degradation via direct binding. Despite its importance to host physiology, very little is known about the role FPN, and iron homeostasis, plays in progression of head and neck cancers. Preliminary work shows that FPN is expressed in normal cells of the oral cavity (oral keratinocytes and gingival fibroblasts). We show repression of FPN and de-regulation of iron homeostasis occurs in advanced (metastatic) head and neck squamous cell carcinoma (HNSCC). Preliminary data also reveals that cell lines with reduced ferroportin levels may make cells more sensitive to ferroptosis. Immunoblots reveal the expression of hepcidin in HNSCC cell lines, indicating that aberrant hepcidin expression may play a role in ferroportin repression in these cell types. The goal of this proposal is to investigate the role FPN repression plays in the progression of HNSCC. We will also probe the interaction of FPN and hepcidin through Small Angle X-ray scattering (SAXS) to shed light on the molecular mechanisms of hepcidin mediated repression of FPN. In aim 1 we will investigate the clinicopathology of FPN repression in HNSCC. We will modulate ferroportin levels in HNSCC cell lines and observe the effect it has on cell proliferation, metastatic potential, and sensitivity to ferroptosis. In aim 2 we will we will explore the role hepcidin plays in the repression of FPN and investigate the allosteric mechanisms of the hepcidin-FPN interaction using SAXS. The findings in this study, despite the outcomes, will be important in characterizing the role iron homeostasis plays in oral health and disease. This fellowship training plan will provide the applicant experience in the fields of cell and cancer biology as well as in the handling and purification of membrane proteins. Training of the applicant will be sponsored by mentors with expertise in the fields of iron biology and cancer biology, as well as in an environment with other trainees.